1. Introduction
Dipeptidyl peptidase IV (DPPIV; EC 3.4.14.5) is a membraneassociated serine-type protease hydrolyzing Xaa-Pro or Xaa-Ala
dipeptides from amino-terminals of oligopeptides at pH 7.0–8.5
(Gossrau, 1979; Gutschmidt and Gossrau, 1981). It participates
in the hydrolysis of a vast number of biologically active peptides, thus altering their activity or receptor specificity (Mentlein,
1999). Besides its protease activity, DPPIV has other functions as a
receptor molecule, co-stimulatory protein and adhesion molecule
(Boonacker and Van Noorden, 2003). Alterations of DPPIV activity
levels have been reported in malignant, autoimmune, inflammatory and infectious diseases (Antczak et al., 2001a,b; Lambeir et al.,
2003). The enzyme has been supposed to be involved in tumor
growth and angiogenesis (Bauvois, 2004). Its up-regulation has
been suggested as an additional indicator for the differentiation
of malignant from benign nodules in thyroid carcinoma (Kholova
et al., 2003). Most of the histological sub-types of lung tumors have
been found as DPPIV-negative (Asada et al., 1993). The DPPIV mRNA

expression has been studied in human bronchial and alveolar cell
lines, including A549 cells, by semiquantitative RT-PCR (Baginski
et al., 2011). The authors have found obvious differences in the
enzyme mRNA levels between different cell lines. DPPIV activity
of A549 cells has been evaluated as much lower than that of primary type II rat alveolar cells (Forbes et al., 1999). However, DPPIV
activity levels have not been thoroughly studied in human lung
cancer cell lines A549 and SK-MES-1 in comparison with a normal
human lung cell line.
The aim of the present work was to compare DPPIV activity in three types of human lung cell lines – P cells (embryonic
diploid cells), A549 (lung adenocarcinoma) and SK-MES-1 (squamous cell carcinoma). For the purpose we used a fluorescent
cytochemical procedure developed on the basis of the fluorogenic substrate 4-(Gly-Pro hydrazido)-N-hexyl-1,8-naphthalimide
(Gly-Pro-HHNI), recently synthesized by us (Ivanov et al., 2009).
The observed differences in the enzyme activity in normal and
tumor cells were estimated by measuring surface and total DPPIV
activities with Gly-Pro-4-nitroanilide substrate (Gly-Pro-pNA).
The decreased levels of the enzyme in tumor cells were readily demonstrated with the fluorogenic substrate, which might
be applied as a cyto-diagnostic tool for non-small lung cell
carcinoma.

of 0.5 mmol substrate (Gly-Pro-HHNI) and 0.5 mg/ml piperonal in
0.1 M phosphate buffer, pH 7.8 for 60 min at 37 ◦ C. After the incubation the sections were post-fixed in 4% neutral formalin for 15 min
at room temperature, stained with haematoxyline consistent with
classical methods of histology and embedded in glycerol/jelly.
2.6. Inhibitor controls
Inhibitor controls were pre-incubated in 0.1 M phosphate buffer
(pH 7.8) containing 0.5 mM inhibitor Phe-Pro-NHONb for 45 min
at room temperature, then transferred to full substrate medium,
supplemented with 0.5 mM of the same inhibitor and allowed to
stain for an hour at 37 ◦ C. Subsequently, the controls were treated
as described above.
All the preparations were observed under OPTON IM 35 fluorescent microscope (Carl Zeiss, Germany) or confocal microscope
Nikon Eclipse Ti-U.
2.7. Surface DPPIV activity of the lung cells

2.3. Cells
P cells (human fetal lung-derived diploid cells), A549 (human
lung adenocarcinoma, ATCC® number: CCL-185TM ) and SK-MES-1
(human lung squamous cell carcinoma, ATCC® number: HTB58TM ) were kindly provided by the National bank for industrial
microorganisms and cell cultures (Sofia, Bulgaria). The tumor cells
were routinely grown in Dulbecco’s Modified Eagle’s Medium
(DMEM), supplemented with 10% Fetal Bovine Serum (FBS) and
antibiotic–antimycotic solution (BioWittaker, Cambrex BioScience,
Belgium) at 37 ◦ C in humidified atmosphere with 5% CO2 . The A549
cells were additionally grown in the same medium but containing
5% FBS or 10% Newborn Calf Serum (NCS). The P cells were cultured
at the same conditions, but the medium was supplemented with
10% NCS. All the cells were grown until 90–95% confluence. For the
cytochemical visualization of DPPIV activity, the cells were grown
on cover slips until 95–100% confluence.
2.4. Tumor tissue
Cryostat sections of tumor tissue extracted at surgery from three
patients with diagnosed squamous cell carcinoma of the lung were
obtained from Pulmonary Clinic “St. Sofia”, Sofia. The protocol was
approved by an independent Ethics Committee. The study was
conducted in compliance with the principles of the Declaration of
Helsinki 1964 and its amendments.
2.5. Cytochemistry and histochemistry
Cells grown on cover slips were washed with PBS and fixed in
paraformaldehyde vapors for 5 min at room temperature. Then,
they were air-dried and covered by celloidin (0.5% celloidin in
absolute ethanol/diethyl ether/acetone 3:3:4) for 30 s at room
temperature. The preparations were incubated in a substrate solution containing 0.3 mM substrate (Gly-Pro-HHNI) and 0.3 mg/ml
piperonal (the substrate and the aldehyde were pre-dissolved in
a minimum amount of dimethylformamide; the incubation solution was filtered before use) in 0.1 M phosphate buffer, pH 7.8 for
an hour at 37 ◦ C. After the incubation, they were post-fixed in 4%
neutral formalin for 15 min at room temperature, stained in 1 !M
Hoechst 33342 aqueous solution for 20 min at room temperature
and embedded in glycerol/jelly (glycerol/15% gelatin 1:1 (Lojda
et al., 1979)).
Cryostat sections of tumor tissue on glass slides were covered
by celloidin (1% celloidin in absolute ethanol/diethyl ether/acetone
3:3:4) for a minute at room temperature. DPPIV activity was localized in the sections incubated in a substrate solution, consisting

The cells were grown in 24-well plates, until they reached
90–95% confluence corresponding to a density of 1 × 105 cells per
well (defined by counting parallel control wells by the Burger’s
camera). Then, the medium was removed, the plates were washed
with 0.1 M phosphate buffer (pH 7.8) and 3 ml solution containing
0.25 mM substrate (Gly-Pro-pNA) in the same buffer was added to
each well. The reaction was carried out at 37 ◦ C and samples were
collected every 30 min. Enzyme reaction was stopped by adding
equal volume of 1.0 M acetate buffer, pH 4.0. The enzyme-catalyzed
release of 4-nitroaniline (pNA) from the substrate was monitored on Ultrospec® 3000 spectrophotometer (Pharmacia Biotech,
Sweden) at 405 nm against a control of substrate solution added to
the cells, collected immediately and blocked with equal volume of
1.0 M acetate buffer, pH 4.0. The results were statistically estimated
by regression analysis and curves showing the time-dependence of
the adsorption at 405 nm were built by means of Sigma Plot 9.0. In
the cases of non-linear correlation, the enzyme activity was determined from the initial rate of the reaction. One unit of enzyme
activity was defined as the amount of enzyme liberating 1 nmol
product (pNA) per minute, per 1 × 105 cells at 37 ◦ C.
2.8. Total DPPIV activity of the lung cells
Cells were grown in 10 cm Petri dishes until they reached
90–95% confluence. Then, the cells were harvested by means of a
rubber policeman and homogenized mechanically in an ice-bath in
0.1 M phosphate buffer (pH 7.8). After a spectrophotometric measurement of protein amount (Layne, 1957), the lysates were diluted
by the same buffer to four varying protein concentrations to final
volume of 1.5 ml. To every dilution, 1.5 ml 0.5 mM Gly-Pro-pNA in
0.1 M phosphate buffer (pH 7.8) was added to obtain a final substrate concentration of 0.25 mM. The reaction was carried out at
37 ◦ C. Aliquots were collected every 60 min and the reaction was
stopped as described above. Absorption of the samples at 405 nm
was measured spectrophotometrically against a control of substrate solution, diluted with equal volume 1.0 M acetate buffer (pH
4.0). One unit of enzyme activity was defined as the amount of
enzyme liberating 1 nmol product (pNA) per minute per 1 mg protein at 37 ◦ C. The results were statistically estimated as described
for the surface DPPIV activity.
3. Results
Recently, we developed a specific fluorogenic substrate (GlyPro-HHNI) for the histochemical localization of DPPIV (Ivanov et al.,
2009). For the purposes of the present experiment we extended

the application of the same substrate for the visualization of the
enzyme activity in cultured cells. Best results were obtained using
cells fixation in paraformaldehyde vapors and embedding the samples in 0.5% celloidin. The cells nuclei stained by Hoechst 33342
were surrounded by a low fluorescent yellow-orange precipitates,
which represented the enzyme activity product (Fig. 1A, D, and G).
Upon excitation by green light (!ex = 540–580 nm) the brilliant red
fluorescence marking DPPIV activity locations could be viewed in

the cells (Fig. 1B, C, E, F, H, and I). The fluorescence intensity of
the final enzyme reaction product was visibly higher in normal
than in tumor lung cells pointing out to a higher DPPIV activity
in the P cell line in comparison to tumor cell lines (Fig. 1A–I).
In the diploid embryonic P cells the reaction product was evenly
deposited in the cells (Fig. 1A, B, and C). This reaction pattern most
probably indicated that the enzyme was uniformly expressed in the
cell periphery. Both tumor cell lines showed a variability between

individual cells. A549 cells were either moderately DPPIV-positive
or DPPIV-negative (Fig. 1D, E, and F). The fluorescent product was
of a homogenous (Fig. 1F) or slightly granular appearance (Fig. 1F
insertion). SK-MES-1 cells displayed a tendency to gather into clusters of moderate or low to none enzyme activity (Fig. 1G, H, and
I). The accumulated fluorescent product was granular probably
indicating intracellular enzyme localization (Fig. 1I). The optical
sectioning upon confocal microscopy confirmed the presence of
cytoplasmic depot of DPPIV in this cell type (Fig. 2). The precise
enzyme locations within SK-MES-1 cells remain to be identified
in the future. The use of inhibitor in different cell lines lead to an
absence of fluorescent reaction product pointing to a total suppression of the enzyme activity (Fig. 1J and K).
Quantitative estimation of DPPIV activity levels in normal and
cancer cell lines were performed with the substrate Gly-Pro-pNA,
containing the same amino acid sequence as the fluorogenic substrate. The activity of the plasma membrane-associated DPPIV and
the total enzyme activity, i.e. the plasmalemma-associated plus
intracellular DPPIV were determined.
Both tumor cell lines showed a linear time-dependence of the
surface enzyme reaction product accumulation during the whole
6 h incubation period (Fig. 3). The reaction rate was estimated as
nearly similar in the two cancer cell lines (Table 1). In the case
of embryonic cells, a non-linear character of the enzyme reaction
product accumulation was detected – it reached a saturation level
showing a high enzyme activity (Fig. 3). The activity of the enzyme
(from the initial rate of the reaction) in the P cells was estimated
to be 8 times higher than this in A549 and 7 times than that of
SK-MES-1 cells (Table 1).
Measurement of the total DPPIV revealed that the relative
enzyme activity was linearly dependant on the protein concentration. In these experiments the time-dependent product
accumulation in A549 cells had a linear character in the range of
used protein concentrations (Fig. 4). On the other hand, SK-MES-1
and P cells displayed a non-linear reaction progress at higher protein concentrations (Fig. 4). The P cell line exhibited the highest
total activity (from the initial rate of the reaction) – the amount of
the released product was ten times higher in comparison to A549
cells and three times as compared to that, released by SK-MES-1
cells (Table 1).
The obtained experimental data about the protein amount per
1 × 105 cells and surface and total enzyme activities were used to
calculate the relative surface activity of DPPIV for the three cell lines
(Table 1).

Fig. 3. The time-course of reaction product accumulation after the substrate GlyPro-pNA (0.25 mM) hydrolysis by plasma membrane-bound DPPIV of different cell
lines. The process was monitored for 6 h at 30 min interval (each result was obtained
on the basis of triplicate experiment).

The use of different types of sera in the culturing medium
of A549 cells did not change the above results for this cell line.
Thus, neither the serum concentration, nor the type of serum used
changed the enzyme activity levels.
The histochemical demonstration of DPPIV activity in tumor
tissues showed that the tumor parenchyma was DPPIV negative
(Fig. 5A and B). Tumor stroma contained lots of DPPIV-positive
cells enclosing the dark zones of carcinoma foci. In the resection
line without tumor infiltrates DPPIV-positive cells were abundant
in all the tissue structures (Fig. 5C and D). In the resection line
with tumor cells infiltrates DPPIV-negative carcinoma foci were
seen surrounded by diffusely distributed enzyme expressing cells
(Fig. 5E and F).

Fig. 4. The time-course of reaction product accumulation after the substrate GlyPro-pNA (0.25 mM) hydrolysis by DPPIV in cell lysates. The process was monitored
for 6 h at 60 min intervals (each result was obtained on the basis of triplicate experiment). The protein quantity in samples was 0.94 mg for cancer cells and 0.66 mg
for P cells.

Relative surface activity of different cell lines.
Relative total activity of different cell lines.

Inhibitor treatment led to a total abolishment of the enzyme
activity, represented by the lack of red fluorescence in the samples
(cultured cells or tissue sections). The inhibitor used is known as
irreversible and specific for DPPIV (De Meester et al., 1992). Thus,
that result proved the specificity of the observed enzyme reaction.
4. Discussion
DPPIV is known to have a soluble and membrane-bound form.
The soluble DPPIV is present in the serum and body fluids (Vanhoof
et al., 1992). The membrane-associated enzyme has a ubiquitous
distribution in the mammalian organs and tissues and is usually

expressed in the apical periphery of epithelial cells (Gossrau, 1985;
Lambeir et al., 2003). The membrane-anchored DPPIV has been
found also in the endosomes of BHK cells (Horstkorte et al., 1996)
and rat hepatocytes (Kreisel et al., 1993) obviously due to the process of internalization-reexpression, as well as in the lysosomes
and trans-Golgi of human hepatocytes (Kyouden et al., 1992; Fukui
et al., 1990). In the human lung, DPPIV is restricted to the endothelial cells of blood vessels, sub-mucosal serous glands and alveolar
epithelial cells, whereas the bronchial epithelium, fibroblasts and
smooth muscles are DPPIV-negative (Van der Velden et al., 1998).
The enzyme activity has been detected also in primary pulmonary
type II cells in culture (Forbes et al., 1999). On the other hand,

Fig. 5. Localization of DPPIV in cryostat sections of squamous lung carcinoma with the substrate Gly-Pro-HHNI. The nuclei are stained with haematoxyline. (A and B) High
enzyme activity in tumor stroma (TS); lack of activity in tumor cells (TC) foci. (C and D) Diffuse DPPIV distribution in all the tissue structures in the resection line without
tumor infiltrates. (E and F) DPPIV-negative tumor cells (TC) foci in the resection line with tumor infiltrates and diffuse enzyme reaction in the surrounding tissue. (A, C, and
E) Light microscopy; (B, D, and F) fluorescent microscopy. Bars = 50 !m.

analyses of DPPIV expression in different histological sub-types of
lung cancer have revealed that some adenocarcinomas express the
enzyme, but large cell carcinomas, small cell cancers, squamous
cell carcinomas and carcinoid tumors lack DPPIV expression (Asada
et al., 1993). Wesley et al. (2004) have reported that non-small cell
lung carcinoma cells express much lower DPPIV than the normal
human lung cells both compared at mRNA and protein levels.
In our experiments, normal fetal lung-derived P cells show a
constantly high DPPIV activity, the enzyme reaction product being
evenly distributed in the cells. The relative surface activity is 95%
and this result is in agreement with the common view that in normal tissues DPPIV is usually most abundant in the plasmalemma.
A549 adenocarcinoma cells show 8–10 times lower surface and
total DPPIV activity than the normal embryonic cells. The observed
varying enzyme activity levels in individual A549 cells could be due
to the high heterogeneity of this cell line. For example, Watanabe
et al. (2002) have shown that A549 cell line consists at least of two
sub-clones exhibiting different susceptibility towards some drugs.
A549 cells are derived from alveolar pneumocytes type II and maintain many of their morphological and biochemical characteristics.
They have specific lamellar bodies and well developed intracellular
membrane system engaged in synthesis, secretion, endocytosis and
recycling of pulmonary surfactant as well as membrane proteins
(Balis et al., 1984). Our results demonstrate that the relative surface
activity in A549 is 93%, which can be explained with the quick externalization of synthesized DPPIV. SK-MES-1 cells also have a low
DPPIV in the cytochemical studies and their surface DPPIV activity
is 7 times lower than that of P lung cells. Their relative surface activity is 35%, indicating that the main quantity of the active enzyme is
restricted within the cells. SK-MES-1 cells have a very low secretion
activity (Finkbeiner et al., 1995) and we suggest that the enzyme
distribution could possibly correlate with disturbances in intracellular membrane transport system. The low secretory activity of the
cells possibly interferes with the enzyme expression on the cell
surface.
Our preliminary experiments on three diagnosed cases of squamous cell lung carcinomas show a high DPPIV activity in the
connective tissue stroma of the carcinomas and lack of enzyme
activity in tumor foci. In our study fluorescent observations are
more demonstrative than light microscopy due to the possibility
of unspecific staining of the histochemical samples. These results
indicate that the possible diagnostic value of DPPIV deserves to be
studied in more details.
In conclusion, our results presented here considered together
with previous studies on other types of lung tumor cells and tissues lead to the reasonable assumption that most lung tumor cells
are deficient in DPPIV activity. The possible therapeutic value of
DPPIV activators remains to be studied in the future. On the other
hand, DPPIV expression levels and localization pattern need to be
analyzed in more details to estimate their application for diagnostic
purposes in human lung cancer.
Acknowledgments
This work is supported by the Bulgarian Ministry of Education
and Science, National Science Fund, Grand nr 1527/05.
References
Antczak, C., deMeester, I., Bauvois, B., 2001a. Ectopeptidases in pathophysiology.
Bioassays 23, 251–260.